This application claims benefit of a prior foreign filing made in France on Aug. 6, 1986, Ser. No. 86-11415.
The present invention relates to the use of an xanthine oxidase (XO) enzyme reaction system in chemiluminescent assays, including immunoassays and DNA probe analysis.
The use of bioluminescence and chemiluminescence in immunoanalysis has been extended to many analytes (KINGLER, W., STRASBURGER C. J., and WOOD W. G., Trends in Analytical Chemistry 2:132-136, 1983; KRICKA L. J., and THORPE G. H. G., Analyst 108:1274-1296, 1983).
Chemiluminescent assays generally fall into two categories:
(a) The quantifiable phenomenon consists of a very brief signal (a few seconds) obtained after instantaneous injection of triggering reagent into the reaction medium. Such is the case with assays using, as a tracer, coupling derivatives of luminol (SCHROEDER H.R., et al: Methods Enzymology 57:424-445, 1978) or of acridinium esters (WEEKS I. and WOODHEAD J. S. in Analytical Applications of Bioluminescence and Chemiluminescence, Academic Press, pp. 185-188, 1984). Apart from the disadvantage associated with the brevity of the signal, this procedure demands the use of an apparatus for injecting the reagents into the measuring chamber itself. In consequence, application to microtitration plates is difficult and relatively sophisticated and expensive apparatus is required for other detection vessels.
(b) The quantifiable phenomenon consists of a longer-lasting signal, which can be measured for a significant time period, enabling measurements to be repeated where appropriate and the samples to be prepared outside the luminometer. In such a system, an injection system is not required, thereby simplifying the design of the luminometer and allowing use of microtitration plates.
At present, only one procedure has been described in which a relatively stable and intense chemiluminescence signal is obtained. This procedure is based on enhancement of the peroxidase-dependent oxidation reaction of cyclic diacylhydrazides by compounds such as synthetic luciferin, 6-hydroxybenzothiazole derivatives or substituted phenols (para-iodophenol) [WHITEHEAD T. P., THORPE G. H. G., CARTER T. J. N. et al., Nature (London) 305:158-159, 1983; THORPE G. H. G., KRICKA L. J. et al., Anal. Biochem. 145:97-100, 1985; THORPE G. H. G., KRICKA L J. et al., Clin. Chem. 31:1335-1341, 1985]. This method is currently applied to the assay of many analytes. However, it should be noted that the term and constancy of the signal is only relative, since the measurements can be performed only during some thirty or forty minutes.
The use of xanthine oxidase in a light producing system has been previously contemplated by others, such as Boguslaski, U.S. Pat. No. 4,363,759 and Molecular Biosystem, Inc., WO-A-85 03,356. However, no evidence or parameters are described which result in a long duration signal. Furthermore, xanthine oxidase is listed together with many other enzyme systems which, as a whole, do not produce both H.sub.2 O.sub.2 and superoxide anion radicals.